Journal of Ethnopharmacology 92 (2004) 23–36

Chemopreventive potential ofAzadirachta indica (Neem) leafextract in murine carcinogenesis model systems

Trisha Dasgupta, S. Banerjee, P.K. Yadava, A.R. Rao∗Cancer Biology and Applied Molecular Biology Laboratories, School of Life Sciences, Jawaharlal Nehru University, New Delhi 110067, India

Received 4 December 2002; received in revised form 5 November 2003; accepted 2 December 2003

Abstract

Numerous laboratory studies reveal that various naturally occurring dietary substances can modify the patho-physiological process ofvarious metabolic disorders and can be an effective preventive strategy for various diseases, including cancer. Indian Neem tree,Azadirachtaindica A. Juss. (family: Meliaceae), contains at least 35 biologically active principles and is widely grown all over the tropics. The effect oftwo different doses (250 and 500 mg per kilogram body weight) of 80% ethanolic extract of the leaves ofAzadirachta indica were examined ondrug metabolizing Phase-I and Phase-II enzymes, antioxidant enzymes, glutathione content, lactate dehydrogenase, and lipid peroxidation inthe liver of 7-week-old Swiss albino mice. Also anticarcinogenic potential ofAzadirachta indica leaf extract was studied adopting protocol ofbenzo(a)pyrene-induced fore-stomach and 7,12-dimethyl benz(a)anthracene (DMBA)-induced skin papillomagenesis. Our primary findingsreveal its potential to induce only the Phase-II enzyme activity associated mainly with carcinogen detoxification in liver of mice. The hepaticglutathione S-transferase (P < 0.005) and DT-diaphorase specific activities (P < 0.01) were elevated above basal level. With reference toantioxidant enzymes the investigated doses were effective in increasing the hepatic glutathione reductase (GR), glutathione peroxidase (GPX),superoxide dismutase (SOD) and catalase (CAT) activities significantly (fromP < 0.005 toP < 0.001). Reduced glutathione measured asnon-protein sulphydryl was found to be significantly elevated in liver (P < 0.005) and in extrahepatic organs (fromP < 0.005 toP < 0.001)examined in our study. Glutathione S-transferase (GST) and DT-diaphorase (DTD) showed a dose-dependent increase in extrahepatic organs.Chemopreventive response was measured by the average number of papillomas per mouse, as well as percentage of tumor-bearing animals.There was a significant inhibition of tumor burden, in both the tumor model system studied (fromP < 0.005 toP < 0.001). Tumor incidencewas also reduced by both the doses ofAzadirachta indica extract.© 2003 Published by Elsevier Ireland Ltd.

Keywords: Cancer chemoprevention;Azadirachta indica; Neem; Drug detoxification enzymes; Antioxidant defense; Skin papillomagenesis; Fore stomachpapillomagenesis

1. Introduction

For the most part of this century, health concerns in thefield of human nutrition that have been centered arounddeficiency disorders of macro and micronutrients withemphasis on the role of essential nutrients in health anddisease. In recent years various dietary constituents havebeen found to provide protection against any disease in-cluding cancer. Any significant role by dietary interventionis encouraging and emerging as an acceptable approach forcontrolling the cancer incidence worldwide (Sporn and Suh,2000; Hakama et al., 1997; Kellof, 2000). It is search andresearch that helps in the identification of such potentialagents, which can either abolish or delay the developmentof carcinogenesis. The latter can be brought about either

∗ Corresponding author. Tel.:+91-11-6166243; fax:+91-11-618-7338.E-mail address: ramesharao2003@yahoo.com (A.R. Rao).

by preventing the activation of carcinogen or by increasingdetoxification or by blocking the interaction of ultimatecarcinogen with cellular macromolecules, or by suppress-ing the clonal expansion of neoplastic cells (Tanaka, 1994;Morse and Stoner, 1996). Azadirachta indica (Indian Neemtree) traditionally employed intensively as folklore remedyfor a wide spectrum of diseases in India (Mulla and Su,1999). Azadirachta indica has a wider arrary of uses thanany other herb. The first recorded use of Neem is attributedto the ancient East Indian ‘Harrappa Culture’ which addedthe plant to dozens of health and beauty aids 4500 yearsago. The centuries old healing system, Ayurvedic medicine,has utilized these timeless Neem formulation as a mainstayof Ayurvedic pharmacy. Its medicinal qualities are outlinedin the earliest ‘Sanskrit’ writings that states uses of variousparts of Azadirachta indica to treat bacterial, fungal, andviral infections and to boost the immune system. Also itsusefulness as a natural non-toxic insecticide among other

0378-8741/$ – see front matter © 2003 Published by Elsevier Ireland Ltd.doi:10.1016/j.jep.2003.12.004

24 T. Dasgupta et al. / Journal of Ethnopharmacology 92 (2004) 23–36

fascinating properties increases it phenomenal applications(Wealth of India, 2000). Numerous scientific reports vali-dates the traditional uses of Neem in both the maintenanceof general health and skin care. Practically every part ofAzadirachta indica (leaves, bark, fruit, flowers, oil, andgum) have been reported to be associated with various reme-dial properties such as, antimicrobial effects (Sai Ram et al.,2000), storage behavior (Sacande et al., 2000), reduction ofparacetamol-induced liver damage (Bhanwara et al., 2000),enhancer of hepatic glutathione and glutathione-dependentenzymes (Arivazhagan et al., 2000), in vitro antiviral ac-tivity (Badam et al., 1999), insecticidal activity (Et-shazlyand et-sharnoub, 2000), antibacterial agent (Das et al.,1999), etc. Chemopreventive potential ofAzadirachta in-dica has also been evaluated recently on 7,12-dimethylbenz(a)anthracene (DMBA)-induced hamster buccal pouchcarcinogenesis (Balasenthil et al., 1999) and against IBDvirus in broilers (Sadekar et al., 1998). The important activeprinciples ofAzadirachta indica are azadirachtin, salanninnimbin, and 6-desacetylnimbin (Mitchell et al., 1997). Theinducibility of drug metabolizing enzyme is one of the re-liable biochemical markers to assess the chemopreventivepotential of a test compound (Prochaska and Fernandes,1993; Banerjee and Rao, 1995). In the present study 80%ethanolic extract of Neem leaf extract was used to evaluatethe induction pattern of these enzymes in the liver of mice,since greatest of the reactions are known to be carried inthe liver. Oxidative stress implicates in all the stages of thedevelopment of cancer as well as in the genesis of otherdiseases so in addition hepatic antioxidant defense enzymescomprising of superoxide dismutase (SOD), catalase (CAT)glutathione peroxidase (GPX), glutathione reductase (GR),and reduced glutathione (GSH) have been evaluated asthey protects the cellular macromolecules against oxida-tive damage. Extrahepatic organs such as lung, kidney, andfore-stomach have also been examined to see the influenceof Neem leaf extract in increasing detoxifying capabilitiesin these organs. Moreover to further substantiate the enthu-siastic biochemical findings, modulatory influence of Neemleaf extract on benzo(a)pyrene-induced fore-stomach andDMBA-induced skin papillomagenesis at peri-initiationallevel was investigated.

2. Materials and methods

2.1. Chemicals

Benzo(a)pyrene [B(a)P], 7,12-dimethylbenzanthracene(DMBA), 1-chloro-2,4-dinitrobenzene (CDNB), 5,5′-dithio-bis-2-nitrobenzoic acid (DTNB), reduced glutathione(GSH), oxidized glutathione (GSSG), pyrogallol, 2,6-dichlo-rophenolindophenol (DCPIP), potassium ferricyanide, tri-ton X-100, ethylenediamine tetracetic acid (EDTA), bovineserum albumin (BSA), sodium pyruvate, thiobarbituricacid (TBA), reduced nicotinamide adenine dinucleotide

(NADH), and reduced nicotinamide adenine dinucleotidephosphate (NADPH) were obtained from Sigma ChemicalCo. (St. Louis, MO, USA). The rest of the chemicals uti-lized were obtained from local firms (India) and were ofhighest purity grade.

2.2. Preparation of modulator

FreshAzadirachta indica (Neem leaf) was obtained fromJNU campus. The leaves were rinsed with water and blotdry. Material of known weight was soxheleted using 80%hydro-alcoholic solvent (80% ethanol, 20% double distilledwater, v/v) three times. Finally the extract was lyophilizedand stored at 4◦C.

2.3. Animals

Random-bred Swiss albino mice (7–8 weeks old) wereused for this study. They were maintained in our air-conditioned animal facility (Jawaharlal Nehru University,New Delhi) with a 12-h light:12-h dark cycle, and provided(unless otherwise stated) with standard food pellets and tapwater ad libitium. All animals were cared for according tothe “Principles of Laboratory Animal care” of the nationalInstitute of Health (NIH, USA) and under strict adherenceto Indian Animal Ethic Committee (IAEC).

2.4. Experiment-I

Modulation of hepatic and extrahepatic carcinogenmetabolising and antioxidant enzymes. Animals were as-sorted in the following group:

Group I (n = 8): Animals were put on a normal dietand treated with 50�l of an emulsion made of peanutoil and double distilled water (ratio 4:1 ml), by oralgavage daily for 15 days; this group of animals servedas negative control.

Group II (n = 8): Animals were put on a normal diet andtreated daily with 250 mg per kilogram body weightof lyophilizedAzadirachta indica (Neem leaf) extract;which was suspended in the control vehicle and wasgiven to the mice 50�l per mouse per day by oralgavage for 15 days.

Group III (n = 8): Animals were put on a normal diet andtreated daily with 500 mg per kilogram body weightof lyophilizedAzadirachta indica (Neem leaf) extract;which was suspended in the control vehicle and wasgiven to the mice 50�l per mouse per day by oralgavage for 15 days.

2.4.1. Preparation of homogenates, cytosol, andmicrosome fractions

Animals were sacrificed by cervical dislocation and theentire liver was then perfused immediately with cold 0.9%NaCl and thereafter carefully removed, trimmed free of ex-

T. Dasgupta et al. / Journal of Ethnopharmacology 92 (2004) 23–36 25

traneous tissue and rinsed in chilled 0.15 M Tris–KCl buffer(0.15 M KCl + 10 mM Tris–HCL, pH 7.4). The liver wasthen blotted dry, weighed quickly, and homogenized in ice-cold 0.15 M Tris–KCl buffer (pH 7.4) to yield 10% (w/v) ho-mogenate. An aliquot of this homogenate (0.5 ml) was usedfor assaying reduced glutathione levels while the remainderwas centrifuged at 10,000 rpm for 20 min. The resultant su-pernatant was transferred into pre-cooled ultracentrifugationtubes and centrifuged at 105,000× g for 60 min in a Beck-man ultracentrifuge (Model-L870M). The supernatant (cy-tosol fraction), after discarding any floating lipid layer andappropriate dilution, was used for the assay of glutathioneS-transferase, DT-diaphorase, lactate dehydrogenase, andantioxidant enzymes, whereas the pellet representing micro-somes was suspended in homogenizing buffer and used forassaying cytochrome P450, cytochrome b5, cytochrome P450reductase, cytochrome b5 reductase, and lipid peroxidation.

2.4.2. Extrahepatic organsThe lung, kidney, and fore-stomach were carefully re-

moved, along with the liver, trimmed free of extraneoustissue and rinsed in chilled 0.15 M Tris–KCl (pH 7.4). Thelung was cut into small pieces. The stomach was openedlongitudinally; the fore-stomach was separated from theglandular stomach and cleaned of all its contents by flush-ing with and changing the buffer five to six times. The lung,kidney, and fore-stomach were then blotted dry, weighedquickly and homogenized in ice-cold 0.15 M Tris–KClbuffer (pH 7.4) to yield a 10% (w/v) homogenate. 0.5 mlaliquot of this homogenate was used for assaying reducedglutathione. The rest of the homogenate was centrifugedat 15,000× g for 30 min at 4◦C; the resulting supernatantobtained was used for assaying glutathione S-transferaseand DT-diaphorase enzymes.

2.4.3. Assay methods

2.4.3.1. Cytochrome P450 and cytochrome b5. Cy-tochrome P450 determined using the carbon monoxidedifference spectra. Both cytochrome P450 and cytochromeb5 content were assayed in the microsomal suspension bythe method ofOmura and Sato (1964), using an absorptioncoefficient of 91 and 185 cm2 M−1 m−1, respectively.

2.4.3.2. NADPH–cytochrome P450 reductase and NADH–cytochrome b5 reductase. Assay of NADPH–cytochromeP450 reductase was done according to the method ofOmuraand Takasue (1970)with some modifications, measuringthe rate of oxidation of NADPH at 340 nm. The reactionmixture contained 0.3 M potassium phosphate buffer (pH7.5), 0.1 mM NADPH, 0.2 mM potassium ferricyanide, andmicrosomal preparation in a final volume of 1 ml. Thereaction started at 25◦C by addition of NADPH. The en-zyme activity was calculated using extinction coefficient6.22 mM−1 cm−1. One unit of enzyme activity is defined asthat causing the oxidation of 1 mol of NADPH per minute.

NADH–cytochrome b5 reductase was assayed accordingto the method ofMihara and Sato (1972), measuring therate of reduction of potassium ferricyanide at 420 nm byNADH. The reaction mixture contained 0.1 M potassiumphosphate buffer (pH 7.5), 0.1 mM NADH, 1 mM potassiumferricyanide, and microsomal preparation in a final volumeof 1 ml. The reaction was started at 25◦C by addition ofNADH. The enzyme activity was calculated using extinctioncoefficient of 1.02 mM−1 cm−1. One unit of enzyme activ-ity is defined as that causing the reduction of one mole offerricyanide per minute.

2.4.3.3. Glutathione S-transferase. The cytosolic as wellas supernatant of extrahepatic glutathione S-transferase ac-tivity was determined spectrophotometrically at 37◦C ac-cording to the procedure ofHabig et al. (1994). The reac-tion mixture (3 ml) contained 1.7 ml of 100 mM phosphatebuffer (pH 6.5), 0.1 ml of 30 mM CDNB, and 0.1 ml of30 mM of reduced glutathione. After preincubating the re-action mixture at 37◦C for 5 min, the reaction was startedby the addition of 0.1 ml diluted cytosol and the absorbancewas followed for 5 min at 340 nm. Reaction mixture with-out the enzyme was used as blank. The specific activityof glutathione S-transferase is expressed as millimoles ofGSH–CDNB conjugate formed per minute per milligram ofprotein using an extinction coefficient of 9.6 mM−1 cm−1.

2.4.3.4. DT-diaphorase. DT-diaphorase activity was mea-sured as described byErnest et al. (1962). With NADH as theelectron donor and 2,6-dichlorophenol-indophenol (DCPIP)as the electron acceptor at 600 nm, the activity was calcu-lated using extinction coefficient 21 mM−1 cm−1. One unitof enzyme activity has been defined as amount of enzymerequired to reduce 1�mol of DCPIP per minute.

2.4.3.5. Reduced glutathione. Reduced glutathione wasestimated as total non-protein sulphydryl group bythe method as described byMoron et al. (1979). Ho-mogenates were immediately precipitated with 0.1 ml of25% trichloroacetic acid and the precipitate was removedafter centrifugation. Free SH groups were assayed in a total3 ml volume by adding 2 ml of 0.6 mM DTNB prepared in0.2 M sodium phosphate buffer (pH 8.0), to 0.1 ml of thesupernatant and absorbance was read at 412 nm using a Shi-madzu UV-160 spectrometer. GSH was used as a standardto calculate millimoles of SH content per gram of tissue.

2.4.3.6. Glutathione reductase. Glutathione reductase wasdetermined by the procedure as described byCarberg andMannervik (1985). Reaction mixture (final volume 1 ml)contained 0.2 M sodium phosphate buffer (pH 7.0), 2 mMEDTA, 1 mM oxidised glutathione (GSSG), and 0.2 mMNADPH. The reaction was started by adding 25�l of cy-tosol and the enzyme activity was measured indirectly bymonitoring the oxidation of NADPH following decrease inOD per minute for minimum 3 min at 340 nm. One unit en-

26 T. Dasgupta et al. / Journal of Ethnopharmacology 92 (2004) 23–36

zyme activity has been defined as nanomoles of NADPHconsumed per minute per milligram of protein based on anextinction coefficient of 6.22 mM−1 cm−1.

2.4.3.7. Glutathione peroxidase. Glutathione peroxidaseactivity was measured by the coupled assay method as de-scribed byPaglia and Valentine (1967). Briefly, 1 ml of thereaction mixture contained 50 mM sodium phosphate buffer(pH 7.0) containing 1 mM EDTA, 0.24 U ml−1 yeast glu-tathione reductase, 0.3 mM glutathione (reduced), 0.2 mMNADPH, 1.5 mM H2O2, and cytosol sample. Reaction wasinitiated by adding NADPH and its oxidation was mon-itored at 340 nm by observing the decrease in OD perminute for 3 min. One unit of enzyme activity have beendefined as nanomoles of NADPH consumed per minute permilligram of protein based on an extinction coefficient of6.22 mM−1 cm−1.

2.4.3.8. Catalase. Catalase was estimated at 240 nm bymonitoring the disappearance of H2O2 as described byAebi(1984). The reaction mixture (1 ml) contained 0.02 ml ofsuitably diluted cytosol in phosphate buffer (50 mM, pH 7.0)and 0.1 ml of 30 mM H2O2 in phosphate buffer. Catalase en-zyme activity has been expressed as moles of H2O2 reducedper minute per milligram of protein.

2.4.3.9. Superoxide dismutase. Superoxide dismutase wasassayed utilizing the technique ofMarklund and Marklund(1974), which involves inhibition of pyrogallol autoxidationat pH 8.0. A single unit of enzyme was defined as the quan-tity of superoxide dismutase required to produce 50% inhi-bition of autoxidation.

2.4.3.10. Lipid peroxidation. Lipid peroxidation in the mi-crosomes was estimated spectrophotometrically by thiobar-bituric acid reactive substances (TBARS) method, as de-scribed byVarshney and Kale (1990), and is expressed interms of malondialdehyde (MDA) formed per milligram ofprotein. In brief, 0.4 ml of microsomal sample was mixedwith 1.6 ml of 0.15 M Tris–KCl buffer to which 0.5 ml of30% TCA was added. Then 0.5 ml of 52 mM TBA wasadded and placed in a water bath for 45 min at 80◦C, cooledin ice and centrifuged at room temperature for 10 min at3000 rpm. The absorbance of the clear supernatant was mea-sured against blank of distilled water at 538.1 nm in spec-trophotometer (Hitachi U-2000).

2.4.3.11. Lactate dehydrogenase. Lactate dehydrogenasewas assayed by measuring the rate of oxidation of NADHat 340 nm, according to the method ofBergmeyer and Bernt(1971). The reaction mixture contains 50 mM potassiumphosphate buffer (pH 7.5), 0.5 mM sodium pyruvate, 0.1 mMNADH, and required amount of cytosolic fraction in finalvolume of 1 ml. The reaction was started at 25◦C by ad-dition of NADH and the rate of oxidation of NADH wasmeasured at 340 nm. The enzyme activity was calculated us-

ing extinction coefficient 6.22 mM−1 cm−1. One unit of en-zyme activity is defined as that which causes the oxidationof 1�mol of NADH per minute.

2.4.3.12. Protein determination. Protein was determinedby the method ofLowry et al. (1951). Using bovine serumalbumin (BSA) as standard at 660 nm.

2.5. Experiment-II

Influence ofAzadirachta indica on DMBA-induced skinpapillomagenesis.

2.5.1. Preparations of chemicals and modulatorDMBA was dissolved in acetone at a concentration of

0.05 mg in 0.01 ml acetone. 250 and 500 mg per kilogrambody weight of Neem leaf extract was prepared in the controlvehicle.

2.5.2. Experimental designThe DMBA-induced skin papillomagenesis was studied

in Swiss albino mice as described byYasukawa et al. (1995),with some modifications. The hairs on the dorsum (2 cmdiameter) of the mice were clipped off 3 days before theapplication of the chemicals, and animals in the resting phaseof hair growth cycle were selected for the experiment. Theanimals were assorted into the following groups:

Group I (n = 15): Animals were treated for 21 daysthrough oral gavage route with the emulsion of peanutoil + double distilled water (4:1 ml), used as vehiclefor feeding the modulator. On 14th day, a single doseof DMBA (0.05 mg/0.1 ml acetone) was applied on theshaven area. Two weeks after the carcinogen applica-tion, 0.1 ml of 1% croton oil in acetone was appliedtwice a week until termination of the experiment. Thisgroup of mice served as positive control group.

Group II (n = 15): All animals within this group weretreated through oral gavage route with Neem leaf ex-tract suspended in the control vehicle at dose level of250 mg per kilogram body weight for 21 days. On 14thday DMBA was topically applied to these animals inthe shaven area (after a gap of minimum 6 h after treat-ment with the modulator) followed by croton oil treat-ment as given to Group I mice.

Group III (n = 15): All animals within this group weretreated through oral gavage route with Neem leaf ex-tract suspended in the control vehicle at dose level of500 mg per kilogram body weight for 21 days. On 14thday DMBA was topically applied to these animals inthe shaven area (after a gap of minimum 6 h after treat-ment with the modulator) followed by croton oil treat-ment as given to Group I mice.

Animals were weighed initially and then every weekly andfinally at autopsy. Papillomas appearing in the shaven areawere recorded at weekly intervals and papillomas >1 mm in

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diameter was included in data analysis only if they persistedfor 2 weeks or more. Animals were sacrificed 20 weeks aftercommencement of the treatments.

2.6. Experiment-III

Influence of Azadirachta indica on benzo(a)pyrene-induced mouse fore-stomach papillomagenesis was studiedas described byAzuine and Bhide (1992). This a modifiedmethod originally described byWattenberg et al. (1980).

2.6.1. Preparations of chemicals and modulatorBenzo(a)pyrene was prepared in peanut oil and the con-

centration adjusted to 1 mg B(a)P/0.1 ml of peanut oil.Lyophilized Neem leaf extract emulsion was prepared inthe control vehicle at two different dose levels, viz. 250 and500 mg per kilogram body weight.

2.6.2. Experimental designThe animals were assorted into the following groups:

Group I (n = 15): Animals were treated for 8 weeksthrough oral gavage route with the emulsion of peanutoil and double distilled water (ratio 4:1 ml), used as ve-hicle for feeding modulator. Two weeks after initiationof vehicle treatment, each mice received 1 mg B(a)Ptwice weekly for 4 weeks. This group of mice servedas positive control group.

Group II (n = 15): Animals were treated with 250 mgper kilogram body weight ofAzadirachta indica (Neemleaf) extract starting 2 weeks before, during and afterthe carcinogen treatment (given as 1 mg B(a)P twiceweekly for 4 weeks) as given to Group I animals.

Group III (n = 15): Animals were treated with 500 mgper kilogram body weight ofAzadirachta indica(Neem leaf) extract starting 2 weeks before, duringand after the carcinogen treatment as given to Group IIanimals.

3. Results

3.1. Hepatic studies

3.1.1. Body and organ weight and general observationsBody weight and relative liver weight at the termination

of experiment have been summarized inTable 1. There is nosignificant difference in the mean body weight in animalstreated with the two different dose of Neem leaf comparedto control. Also there was no alteration in the liver-somaticindex, and in the microsomal and cytosolic protein valuesbetween the control and modulator treated animals. The oraladministration of Neem leaf extract did not cause any appar-ent clinical signs such as survivability, or any gross visiblechanges attributable to toxicity in the liver, lung, and kidneyof mice.

3.2. Lactate dehydrogenase and lipid peroxidation

The activity of LDH was diminished by 0.74-folds (P <

0.001) and 0.59-folds (P < 0.005) following low and highdoses of Neem leaf extract treatment. Level of lipid perox-idation was inhibited by both the lower and higher dose ofNeem leaf treatment to 0.82- and 0.90-folds, respectively;though the changes were not significant.

3.3. Phase I enzymes

Cytochrome P450 revealed no significant alterations fromits basal constitutive activities. Cytochrome b5 reduced sig-nificantly by 0.75-folds (P < 0.005) and 0.78-folds (P <

0.005) following low and high dose of Neem leaf extracttreatment. Cytochrome P450reductase and cytochrome b5 re-ductase showed significant reduction. By the low dose therewas a decrease of 0.85-folds (P < 0.05) and 0.93-folds inthe specific activity of cytochrome P450 reductase and cy-tochrome b5 reductase. Whereas the high dose of Neem leaftreatment reduced their activity by 0.80 (P < 0.001) and0.68 (P < 0.01), respectively.

3.4. Phase II enzymes

Specific activities of both the phase II enzymes stud-ies, viz. glutathione S-transferase and DT-diaphorase (DTD)have shown significant increase at both the dose levels oftreatment with Neem leaf extract with respect to the controlgroup.

3.4.1. Glutathione S-transferaseGlutathione S-transferase enzyme activity was enhanced

by 1.66-folds (P < 0.005) and 1.6-folds (P < 0.005) in lowand high-dose groups, respectively.

3.4.2. DT-diaphoraseSpecific activity of DTD was increased by 1.16-folds (P <

0.01) and 1.12-folds in low and high doses, respectively.

3.5. Antioxidant parameters

3.5.1. Superoxide dismutaseThe hepatic superoxide dismutase activity was increased

by 1.23-folds (P < 0.005) and 1.22-folds (P < 0.005) inlow and high doses relative to control.

3.5.2. Glutathione peroxidaseAn induction of 1.6-folds (P < 0.001) and 1.63-folds

(P < 0.005) was evident following the treatment by the lowand high dose of Neem leaf extract.

3.5.3. CatalaseCatalase activity was increased in both low and high doses

by 1.43-folds (P < 0.005) and 1.63-folds (P < 0.005) rela-tive to control.

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Table 1Modulatory influence of the two different doses ofAzadirachta indica on weight gain profiles, protein levels and toxicity related parameters

Groups and treatment Body weight (g) Liver weight×100/final body weight

LDHa Protein (mg ml−1)

Initial Final Microsome Cytosol

Control vehicle 31.30± 1.28 (100) 34.00± 0.92 (100) 6.45± 0.51 (100) 2.02± 0.16 (100) 7.03± 1.47 (100) 9.44± 1.99 (100)250 mg per kilogram body weight of Neem leaf 35.13± 3.22 (112.23) 38.12± 2.35 (112.11) 6.75± 0.87 (104.65) 1.50± 0.16∗∗ (74.25) 7.60± 1.87 (108.10) 9.60± 1.51 (101.70)500 mg per kilogram body weight of Neem leaf 32.00± 4.37 (102.23) 37.75± 2.71 (111.02) 7.71± 0.77 (119.53) 1.20± 0.20∗ (59.40) 8.36± 1.52 (118.91) 8.99± 0.88 (95.23)

Values are expressed as mean± S.D. of six to eight animals. Values in parentheses represent relative changes in parameters assessed (i.e. levels of parameter assessed in livers of micereceiving testsubstance to that of control mice). Abbreviation: LDH, lactate dehydrogenase. Treatment duration: 15 days.

a Micromole per milligram of protein.∗ Significant changes against vehicle treated negative control (P < 0.005).∗∗ Significant changes against vehicle treated negative control (P < 0.001).

T. Dasgupta et al. / Journal of Ethnopharmacology 92 (2004) 23–36 29

3.5.4. Glutathione reductaseLow and high doses of Neem leaf extract augmented the

GR activity by 1.2-folds (P < 0.005) and 1.22-folds (P <

0.005) when compared to control group.

3.5.5. Reduced glutathioneThe level of GSH, the non-enzymatic antioxidant protein

was enhanced by 1.75-folds (P < 0.005) and 1.22-folds(P < 0.005) by the low and high doses, respectively.

3.6. Extrahepatic studies

3.6.1. Reduced glutathioneIn the fore-stomach the activity of GSH was increased by

1.85-folds (P < 0.005) and 2-folds (P < 0.001). In kidneythere was no significant increase at the low dose level but asignificant increase of 1.36-folds (P < 0.001) was revealedat the high dose of treatment relative to control. In lung GSHactivity was augmented by 1.64-folds (P < 0.005) and 1.75-folds (P < 0.005) by the low and high doses of Neem leafextract pretreatment.

3.6.2. DT-diaphoraseRelative to control animals the specific enzyme activity

was maximally induced in the fore-stomach being 1.22-folds(P < 0.001) and 1.27-folds (P < 0.001) by the low and highdoses, respectively. In kidneys of the treated mice a dose-dependent increase of 1.25-folds (P < 0.005) and 1.28-folds(P < 0.001) was evident. In the lung an induction of 1.31-folds (P < 0.005) and 1.37-folds (P < 0.005) above basalactivity was noted following the low and high dose of Neemleaf extract treatment.

3.6.3. Glutathione S-transferaseIn the fore-stomach GST activity was increased by 1.42-

folds (P < 0.005) and 1.26-folds (P < 0.005) by low andhigh doses of Neem leaf treatment, respectively. In kidneysof the treated mice both the group had the same increase of

0

10

20

30

40

50

60

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16Weeks

Nu

mb

er o

f tu

mo

rs

Control Low dose High dose

C

A

Fig. 1. Effect of two different doses of Neem leaf (Azadirachta indica) extract on DMBA-induced skin papillomagenesis. Control DMBA+ 250 mg perkilogram body weight of Neem leaf extract, high-dose DMBA+ croton oil+ 500 mg per kilogram body weight of Neem leaf extract. Effective no: 18animals per group.C(P < 0.005) andA(P<0.01) represent significant changes against the control.

1.43-folds (P < 0.005) relative to control group. In lung, adose-dependent increase of GST activity was noticed, being1.25-folds (P < 0.01) and 1.26-folds (P < 0.005) by thelow and high dose of Neem treatment.

3.7. Mouse skin papillomagenesis experiment

Tables 2–5and Fig. 1 depict results of skin papilloma-genesis obtained from treatment of Neem leaf during peri-initiational period. No adverse effect on body weight gain,during the observation period noticeable. Moreover no evi-dence of development of spontaneous tumor including skinlesions in our colony of Swiss mice has been encountered. Inthe DMBA-induced skin papillomagenesis study the meannumber of papillomas per animal (tumor burden) in controlanimals was 5.00± 2.42, whereas it was 2.41± 2.0 (P <

0.005), and 3.12± 2.33 (P < 0.05) in the lower and higherdose treated animals, respectively. The tumor incidence wasonly 64.7 and 68.75% (35.3 and 31.25% reduction) in lowand high dose of Neem leaf treated groups against the 100%tumor incidence of the control group.

3.8. Mouse fore-stomach papillomagenesis experiment

Table 6depicts the result obtained by Neem leaf extractsupplementation on benzo(a)pyrene-induced fore-stomachpapillomagenesis. No difference was noticeable in weightgain profile of animals treated with either doses of Neem leafextract as well as in the positive control group of mice. Hun-dred percent of the control animals developed fore-stomachpapilloma by benzo(a)pyrene treatment. The mean numberof papilloma per mouse (tumor burden) in this group of an-imals was 3.73 ± 1.58. In contrast animals pre and posttreated with 250 and 500 mg per body weight of Neem leafextract, was decreased to 1.44± 1.58 (P < 0.001) in lowdose and 1.73±1.33 (P < 0.005) in high dose, respectively.Also tumor incidence was only 61% (39% reduction) and73% (27% reduction) in low and high dose of Neem leaf-

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Table 2Modulatory influence of two different doses ofAzadirachta indica on mice hepatic phase I and phase II drug metabolizing enzymes

Groups Cyt P450a Cyt b5

a Cyt P450b Cyt b5 Rc GSTd DTDe

Control vehicle 0.34± 0.05 (100) 0.28± 0.01 (100) 0.21± 0.02 (100) 2.06± 0.31 (100) 3.19± 0.16 (100) 0.050± 0.005 (100)250 mg per kilogram body weight of Neem leaf 0.31± 0.04 (91.17) 0.21± 0.00∗∗∗ (75.00) 0.18± 0.016∗ (85.71) 1.92± 0.22 (93.20) 5.29± 0.90∗∗∗ (165.83) 0.058± 0.002∗∗ (116)500 mg per kilogram body weight of Neem leaf 0.28± 0.04 (82.35) 0.22± 0.02∗∗∗ (78.57) 0.17± 0.01∗∗∗∗ (80.95) 1.42± 0.23∗∗ (68.93) 5.12± 0.78∗∗∗ (160.50) 0.056± 0.002 (112)

Values are expressed as mean± S.D. of six to eight animals. Values in parentheses represent relative change in parameters assessed (i.e. levels of activity in livers of mice receiving test substanceto activity in liver of control mice). Abbreviations: Cyt P450, cytochrome P450; Cyt b5, cytochrome b5; Cyt P450 R, cytochrome P450 reductase; Cyt b5 R, cytochrome b5 reductase; GST, glutathioneS-transferase; DTD, DT-diaphorase. Treatment duration: 15 days.

a Nanomole per milligram of protein.b Micromole of NADPH oxidized per minute per milligram of protein.c Micromole of NADH oxidized per minute per milligram of protein.d Micromole of CDNB–GSH conjugate formed per minute per milligram of protein.e Micromole of DCPIP reduced per minute per milligram of protein.∗ Significant changes against control (P < 0.05).∗∗ Significant changes against control (P < 0.01).∗∗∗ Significant changes against control (P < 0.005).∗∗∗∗ Significant changes against control (P < 0.001)

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Table 3Modulatory influence of two different doses ofAzadirachta indica on mice hepatic antioxidant related parameters and lipid peroxidation

Groups GSHa SODa CATb GPXc GRd LPe

Control vehicle 2.85± 0.25 (100) 11.93± 1.23 (100) 59.10± 11.49 (100) 20.53± 1.86 (100) 33.60± 2.80 (100) 1.07± 0.10 (100)250 mg per kilogram body weight of Neem leaf 5.00± 0.61∗ (175.43) 14.67± 1.64∗ (122.96) 84.40± 2.17∗ (142.80) 33.00± 2.55∗∗ (160.74) 40.32± 3.89∗ (120.0) 0.88± 0.15 (82.24)500 mg per kilogram body weight of Neem leaf 4.01± 0.68∗ (140.70) 14.52± 0.98∗ (121.70) 96.17± 5.20∗ (162.72) 33.55± 2.47∗ (163.41) 41.00± 2.77∗ (122.02) 0.97±.0.08 (90.65)

Values are expressed as mean± S.D. of six to eight animals. Values in parentheses represent relative change in parameters assessed (i.e. levels of activity in livers of mice receiving test substance toactivity in liver of control mice). Abbreviations: GSH, reduced glutathione; GPX, glutathione peroxidase; GR, glutathione reductase; SOD, superoxide dismutase; CAT, catalase; LP, lipid peroxidation.Treatment duration: 15 days.

a Nanomole GSH per gram of tissue.b Specific activity expressed as micromole per milligram of protein.c Micromole H2O2 consumed per minute per milligram of protein.d Nanomole of NADPH consumed per minute per milligram of protein.e Nanomole of malondialdehyde formed per milligram of protein.∗ Significant changes against control (P < 0.005).∗∗ Significant changes against control (P < 0.001).

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Table 4Modulatory influence ofAzadirachta indica on detoxifying and antioxidant enzyme profiles in the extrahepatic organs of mice

Groups and treatment Organ weight×100/final body weight

GSHa GSTb DTDc Protein (mg ml−1)

LungControl vehicle 0.79± 0.15 (100) 0.97± 0.13 (100) 0.49± 0.04 (100) 0.029± 0.003 (100) 6.56± 1.59 (100)Neem extract (250 mg per kilogram body weight) 0.73± 0.12 (92.40) 1.60± 0.19∗∗ (164.94) 0.61± 0.06∗ (124.48) 0.038± 0.005∗∗ (131.03) 6.18± 1.37 (94.20)Neem extract (500 mg per kilogram body weight ) 0.85± 0.13 (107.59) 1.70± 0.16∗∗ (175.25) 0.62± 0.06∗∗ (126.53) 0.040± 0.005∗∗ (137.93) 5.99± 1.35 (91.31)

KidneyControl vehicle 1.83± 0.12 (100) 1.17± 0.20 (100) 0.39± 0.05 (100) 0.039± 0.003 (100) 7.55± 1.24 (100)Neem extract (250 mg per kilogram body weight ) 1.97± 0.19 (107.65) 1.32± 0.19 (112.82) 0.56± 0.05∗∗ (143.58) 0.049± 0.002∗∗ (125.64) 6.60± 1.08 (87.41)Neem extract (500 mg per kilogram body weight ) 2.22± 0.33 (121.31) 1.60± 0.17∗∗∗ (136.75) 0.56± 0.05∗∗ (143.58) 0.050± 0.006∗∗∗ (128.20) 6.16± 0.96 (81.58)

Fore-stomachControl vehicle 0.21± 0.03 (100) 0.77± 0.10 (100) 0.26± 0.04 (100) 0.022± 0.002 (100) 5.66± 0.96 (100)Neem extract (250 mg per kilogram body weight ) 0.24± 0.04 (114.28) 1.43± 0.25∗∗ (185.71) 0.37± 0.02∗∗ (142.30) 0.027± 0.002∗∗∗ (122.72) 5.20± 0.55 (91.87)Neem extract (500 mg per kilogram body weight ) 0.25± 0.04 (119.04) 1.54± 0.17∗∗∗ (200.00) 0.33± 0.02∗∗ (126.92) 0.028± 0.002∗∗∗ (127.27) 5.30± 1.02 (93.63)

Values are expressed as mean± S.D. of six to eight animals. Values in parentheses represent relative change in parameters assessed (i.e. levels of activity in extrahepatic organs of mice receiving testsubstance to activity in extrahepatic organs of control mice). Abbreviations: GSH, reduced glutathione; GST, glutathione S-transferase; DTD, DT-diaphorase. Treatment duration: 15 days.

a Nanomole of GSH per gram of tissue.b Micromole CDNB-GSH conjugate formed per minute per milligram of protein.c Micromole of DCPIP reduced per minute per milligram of protein.∗ Significant changes against control (P < 0.01).∗∗ Significant changes against control (P < 0.005).∗∗∗ Significant changes against control (P < 0.001).

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Table 5Effect of two different doses ofAzadirachta indica on DMBA-induced skin papillomagenesis in mice

Groups and treatment Body weight (g) Total tumorincidence(%)

Tumor burden(tumor/mouse)

Percent inhibitionof tumor burden

Initial Final

Only vehicle+ DMBA+ croton oil

22.55± 2.12 31.73± 3.43 100 5.00± 2.42 –

Neem leaf extract(250 mg/kg b.weight)+ DMBA + croton oil

27.38± 3.07 34.73± 3.43 64.70 2.41± 2.0∗ 51.76

Neem leaf extract (500 mgper kilogram body weight )+ DMBA + croton oil

25.72± 2.30 33.66± 2.14 68.75 3.12± 2.33∗∗ 37.50

Values are expressed as mean± S.D. of 12–15 animals.∗ Significant changes against control (P < 0.005).∗∗ Significant changes against control (P < 0.01).

Table 6Effect of two different doses ofAzadirachta indica on benzo(a)pyrene [B(a)P]-induced fore-stomach papillomagenesis in mice

Groups and treatment Body weight (g) Total tumorincidence (%)

Tumor burden(tumor per mouse)

Percent inhibitionof tumor burden

Initial Final

Benzo(a)pyrene+ only vehicle 20.75± 2.13 24.62± 2.20 100 3.73± 1.58 –Benzo(a)pyrene+ Neem

leaf extract (250 mg perkilogram body weight )

20.45± 1.92 25.84± 3.70 61 1.44± 1.58∗ 61.32

Benzo(a)pyrene+ Neemleaf extract (500 mg perkilogram body weight)

20.62± 2.23 27.87± 1.92 73 1.73± 1.33∗∗ 53.58

Values are expressed as mean± S.D. of 12–15 animals.∗ Significant changes against control (P < 0.001).∗∗ Significant changes against control (P < 0.005).

treated animals in contrast to the 100% tumor incidence ofcontrol group.

4. Discussion and conclusion

It is only from the last half of the past century that foodsare being scientifically investigated and evaluated for theirdisease prevention and therapeutic effects. Interest in evalu-ating oriental medicinal herbs and edible phytoproducts foruse in cancer preventive strategies is gaining grounds forprioritization in public health programs of many develop-ing as well as developed countries (Ren and Liece, 1997;Pezzulto, 1995; Chemoprevention Working Group, 1999).Several experimental evidence for all common cancer siteshave indicated that intake of fruits and vegetables and anumber of other dietary items are associated with decreasedcancer incidence (Murakami et al., 1996; Morse and Stoner,1996; Stewart et al., 1996). The present experiment was de-signed to study the inducing potential of Neem leaf on phaseI, phase II, and antioxidant enzymes and also to check itsefficacy against benzo(a)pyrene and DMBA-induced fore-stomach and skin papillomagenesis.

From almost the very beginning of recorded human his-tory people have taken advantage of the remarkable Neemtree, even today rural Indians refer to the Neem tree as their

“Village Pharmacy” due to its increadible arrary of healingproperties ranging from bad teeth and bedbugs to ulcers andmalaria (The wealth of India, 2000). Results obtained fromthe present study testify Neem leaf administration to miceaffects liver enzyme activities correlated with attenuating therisk of chemical carcinogenesis.

Cytochrome P450 (CYP) isoenzymes, are essential for ini-tiating conversion of lipophilic xenobiotics/carcinogens intomore polar, hydrophilic, water-soluble metabolites. At thesame time, induction of phase I enzymes is considered a po-tential cancer risk factor due to the activation of carcinogensto ultimate carcinogens (Lampe et al., 2000). Neem leaf ex-tract pretreatment was ineffective in elevating the basal levelactivity of hepatic phase I enzymes.

Phase-II detoxifying enzymes are a class of widely dis-tributed enzymes that detoxify carcinogens either by de-stroying their reactive centers or by conjugating them withendogenous ligands facilitating their excretion. GlutathioneS-transferase and DT-diaphorase are two major phase IIenzymes. GST catalyses the conjugation of a variety of en-dogenous and exogenous compounds with the non-proteinthiol, glutathione. This reaction inhibits reactive elec-trophiles from reaching cellular targets and results in theproduction of a thio-ether linked glutathionyl conjugate thatis less cytotoxic. There are strong evidences to suggest therelationship between the depletion of GST and increase in

34 T. Dasgupta et al. / Journal of Ethnopharmacology 92 (2004) 23–36

cancer susceptibility (Abraham and Singh, 1999). There areevidences to show that GST activity is elevated to cell linesresistant to chemotherapeutic agents (Hayes and Pulford,1995). Glutathione S-transferase predominantly participatesin detoxification of xenobiotics, and the ability to induceglutathione S-transferase is a property found in many ofthe chemopreventive agents ameliorating toxicity and car-cinogenicity (Wilkinson and Clapper, 1997; Kenster, 1995).Moreover many of the naturally occurring chemopreventiveagents have been reported to convert the DNA damagingentities into excretable metabolites through induction ofGST (Hu and Singh, 1997; Coles and Ketterer, 1990).

DT-diaphorase is a flavoprotein that catalyzes the two-electron reduction of quinones and other nitrogen oxides.This reaction prevents one electron redox cycling of thesegroups, thereby preventing the formation of DNA damagingreactive oxygen species. Reduction of quinones and nitrogenoxides might also make them available for conjugation withUDP–glucuronic acid, facilitating their excretion (Begleiteret al., 1997; Benson et al., 1980; Talalay et al., 1995). Neemleaf extract increased the activities of both these phase IIenzymes in the liver and in all the extrahepatic organs ex-amined, viz. fore-stomach, liver, and lungs in the presentstudies.

Reference to antioxidant enzyme status in liver, the spe-cific activity of almost all the antioxidant related enzymesincluding glutathione peroxidase, glutathione reductase,catalase, and superoxide dismutase were found elevatedabove the control basal values either in dose-dependant ordose-independent manner. Superoxide dismutase plays animportant role in catalyzing the dismutation of superoxideradicals. Increase in SOD activity should accelerate theremoval of the reactive oxygen species. Catalase, whoseactivity has also been augmented by the Neem extract,helps in removing the hydrogen peroxide produced by theaction of SOD. Induced SOD activity along with that ofcatalase explains the decrease in lipid peroxidation, whichis an indicator of oxidative stress that persists in the cell.Glutathione reductase, another major antioxidant enzymecatalyzes the NADPH-dependent reduction of glutathionedisulfide to glutathione thus maintaining the GSH level inthe cell (Mayes and McLellan, 1999). Neem leaf extracttreatment has significantly increased the activity of GRthere by helping the cell to maintain the basal level ofGSH, which is important for many other GSH-dependentdetoxification reactions (Meister and Glutathione, 1994).

The activity of lactate dehydrogenase, which is an indica-tor of cell damage; decreased significantly at both the doselevels of Neem leaf extract implying the cytoprotective ef-fect exerted byAzadirachta indica. Apart from the numerousfunctions like transport of ions/sugars, synthesis of proteinand DNA, maintenance of membrane integrity, etc. reducedGSH participates in spontaneous scavenging of electrophilesor free radicals produced in reactions catalyzed by GPX andGST (Vander Laarse, 1995). GSH activity was increased inabove basal level in the liver as well as all the extrahepatic

organs investigated. Thus, the induction of GSH by Neemleaf extract facilitates the protection of the cell against freeradical-induced damage.

Benzo(a)pyrene is a polycyclic aromatic hydrocarbon andis proved to be an effective carcinogen in producing lungand fore-stomach neoplasia, in many animal model sys-tems. Since B(a)P is a widespread environmental pollutantand is believed to be a risk factor in human chemical car-cinogenesis, it becomes increasingly important to identifythe naturally occurring/synthetic compounds that can inter-fere with B(a)P-induced carcinogenesis. There are reportsto show that there are a few substances that can inhibit thisprocess (Katiyar et al., 1993; Badary et al., 1999). So an-other objective of our present study was to see the efficacyof Azadirachta indica to inhibit the incidence of papillo-magenesis in mouse skin and fore-stomach after carcinogeninitiator and/or promotor was given to appropriate animalmodels. There was a reduction in the tumor incidence aswell as in tumor burden at both the sites of the animal mod-els that we used.

Numerous reports strongly support the correlation be-tween the induction of phase I, phase II, and antioxidantenzymes and cancer incidence (Dasgupta et al., 2001; Mccord, 1993; Wilkinson and Clapper, 1997). In the presentwork administration of Neem leaf extract has shown a sig-nificant induction of mainly phase II enzymes and antiox-idative parameters, along with a significant decrease in thespecific activity of LDH and the level of lipid peroxidation.These effects when considered together presumably resultedin enhanced carcinogen detoxification byAzadirachta indicaand its blocking/suppressing effect on “initiation” stages ofcarcinogenicity.

In conclusion we may say that for thousand of yearshumans have sought to fortify their health and cure var-ious ills with herbal remedies. Throughout this time thesearch for a true panacea or cure-all has been undertaken.While hundreds of substances have been tried and testedfew have withstood mordern scintific scrutiny. Perhaps noother herbal medicinal plant meets the true defination ofa panacea than Indian Neem tree. Every part of this fas-cinating plant has been used to treat hundreds of differ-ent maladies from antient to mordern times. In the presentstudy we have evaluated the carcinogen-induced stomachand skin cancer chemoprevention potential ofAzadirachtaindica. Results obtained strongly suggest thatAzadirachtaindica can significantly inhibit the chemical carcinogenesisat peri-initiational stages of carcinogenesis though modu-lation of phase II detoxification enzymes, elevation of an-tioxidant enzymes level and by inhibiting lipid peroxidationand lactate dehydrogenase-induced damages. In our studythe low dose of Neem leaf was more potent in inhibitingthe cancer incidence than the high dose. SinceAzadirachtaindica has not shown any toxic effect at the present givendose levels it could well be applied in cancer chemopre-vention to reduce the risk of cancer and to combat cancerburden.

T. Dasgupta et al. / Journal of Ethnopharmacology 92 (2004) 23–36 35

Acknowledgements

Trisha Dasgupta is thankful to Indian Council for CulturalRelationship (ICCR), Government of India for providing thePh.D. scholarship.

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